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Biochemical and Biophysical Research Communications xxx (2018) 1e6
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The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition Feng Ke a, Jinqiang Yu a, Wei Chen b, Xiaomin Si c, Xinhui Li d, Fang Yang a, Yingying Liao e, *, Zhigang Zuo f, ** a
Department of Ophthalmology, Renmin Hospital, Hubei University of Medicine, Shiyan, China Department of Critical Care Medicine, Taihe Hospital, Hubei University of Medicine, Hubei, China Department of Cardiopulmonary Rehabilitation, Renmin Hospital, Hubei University of Medicine, Shiyan, China d Department of Oncology, Shiyan Taihe Hospital, Shiyan, China e Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Shiyan, China f Department of Oncology, Renmin Hospital, Hubei University of Medicine, Shiyan, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2018 Accepted 10 June 2018 Available online xxx
Mitochondria has been identified as a promising target in several cancers. However, little is known on the effects of targeting mitochondria in retinoblastoma. In this work, we show that anti-malarial atovaquone, at clinically achievable concentration, demonstrates inhibitory effects to retinoblastoma cells, to a more extent than in normal retinal cells. Atovaquone also significantly increases chemosensitivity in retinoblastoma. Importantly, we show that retinoblastoma cells have higher level of mitochondrial respiration, membrane potential, mass and ATP compared to normal retinal cells. Although atovaquone significantly inhibits mitochondrial respiration and decrease ATP level in both malignant and normal retinal cells in a similar manner, atovaquone induces much more oxidative stress and damage in retinoblastoma than normal retinal cells. These suggest that normal retinal cells are more tolerable to mitochondrial dysfunctions than retinoblastoma cells. We further demonstrate that atovaquone targets Akt/AMPK/mTOR signaling via inducing mitochondrial dysfunction. Our pre-clinical work demonstrates the translational potential of atovaquone as an addition to the treatment armamentarium for retinoblastoma. Our work also demonstrates the differences of mitochondrial biogenesis and function in malignant versus normal retinal cells which are important for the targeted therapy in retinoblastoma. © 2018 Elsevier Inc. All rights reserved.
Keywords: Atovaquone Retinoblastoma Normal retina Mitochondria Akt/AMPK/mTOR
1. Introduction Retinoblastoma is an ocular cancer of retinal origin and typically occurs children younger than 5 years. Although the standard treatment including enucleation, chemotherapy and radiation significantly improves clinical outcome, the management of retinoblastoma is still challenge in developing countries [1]. Mutation of the tumor suppressor gene retinoblastoma 1 (RB1) plays a
* Corresponding author. Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Maojian District, Chaoyangzhong Road 39, 442000, China. ** Corresponding author. Department of Oncology, Renmin Hospital, Hubei University of Medicine, Maojian District, Chaoyangzhong Road 39, 442000, China. E-mail addresses: [email protected] (Y. Liao), [email protected] (Z. Zuo).
central role in retinoblastoma transformation and progression [2,3]. Substantial evidence has shown extensive heterogeneity in retinoblastoma at molecular, cellular and tumor level [4], suggesting that targeting common cancer drivers may represent a potential therapeutic strategy for retinoblastoma. The mitochondria is an important energy and biosynthetic factories supporting cancer cell growth and survival [5]. Apart from energy metabolism, other functions of mitochondria include calcium homeostasis, redox regulation and apoptosis [6]. Importantly, studies have recently shown that cancer cells have increased mitochondrial biogenesis and rely more on mitochondrial respiration compared to normal cells [7e9]. Inhibition of mitochondrial functions display anti-cancer activities while having less toxicity to normal counterparts [10]. Targeting mitochondria using clinically available drugs as a cancer therapeutic strategy has attracted much
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Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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attention in the recent years and shown translational potential in pre-clinical and clinical settings [8,11]. Atovaquone is an anti-malarial drug with its target on cytochrome bc1 complex and therefore affecting mitochondrial function [12,13]. In this study, we investigated the effect and target of atovaquone in retinoblastoma (malignant) and normal retinal cells. We further investigated the basal mitochondrial biogenesis of malignant and normal retinal cells to understand their differential response to atovaquone. Our findings demonstrate that inhibition of mitochondrial respiration as a potential targeted strategy for retinoblastoma. 2. Materials and methods 2.1. Primary cells, cell culture and generation of r0 cell line Human retinoblastoma cell lines RB116 (Kerafast Inc. US), Y79 and WERI-Rb-1 (ATCC, US) and immortalized normal retinoblastoma pigmented epithelial cell line (RPE-1, ATCC, US) were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, and 4.5 g/L glucose. Primary normal retinoblastoma pigmented epithelial cell (HNRPE, Abm, US) was grown in extracellular matrix (Abm, US)-coated flask using the media (Abm, Cat. No. TM4057) according to manufacture's instruction. Mitochondria DNA-deficient r0 cell line was established according to the method previously described [14]. The lack of mitochondrial DNA in r0 cells was confirmed using reverse transcriptionePCR (data not shown).
2.2. Migration assays Migration assay was performed using the Boyden chamber which consists a Falcon cell culture insert and 24-well plate chamber. Cells (10,000/well) and atovaquone were added into the cell culture insert. 20% FBS was placed into the lower chamber. The assembled cell culture insert chamber was incubated for 6e8 h. Cells on the upper surface of the insert were then removed with a cotton swab. Migratory cells on the lower surface of inserts were fixed with 4% formaldehyde (Sigma, US) for 10 min, then stained with 0.4% Giemsa and counted under using light microscope (Zeiss, Germany). 2.3. Proliferation assay Cells (10, 000/well) were treated with atovaquone, vincristine or combination for 72 h in 96-well-plate. Proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation assay kit (Promega, US). 2.4. Mito stress and glycolytic stress test assays Cells (10,000/well) were seeded, cultured and treated with atovaquone in XF96 cell culture plates. After treatment, media were replaced by XF assay medium (Seahorse Bioscience, US) and incubated at 37 C in a CO2-free environment for 1 h. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at 37 C using an XF96 extracellular analyzer
Fig. 1. Atovaquone selectively targets retinoblastoma cells and enhances the inhibitory effects of chemotherapeutic agent. Atovaquone is more effective in inhibiting proliferation (A) and inducing apoptosis (B) in retinoblastoma cells: RB116, Y79 and WERI-Rb1 cells than normal retinal cells: RPE-1 and HNRPE. Atovaquone significantly enhances the anti-proliferative (C) and pro-apoptotic (D) effects of vincristine in retinoblastoma cells. Atovaquone at 5 mM was used in combination studies. *p < 0.05, compared to control or vincristine alone.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
F. Ke et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e6
(Seahorse Bioscience, US) as per the manufacture's instructions. All injection reagents (as detailed in the figure legends) were adjusted to pH 7.4. The Seahorse software calculated OCR and ECAR automatically. 2.5. Flow cytometry Cells (50, 000/well) were treated with atovaquone in 24-wellplate for 24 h. Cells were stained with CM-H2DCFDA (indicates intracellular ROS), MitoSox Red (indicates mitochondrial superoxide), MitoTracker (indicates mitochondrial mass), or 5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethyl benzimidazolylcarbocyanine iodide (JC-1 to indicate mitochondrial membrane potential) as per manufacture's instructions (Invitrogen, US). Apoptosis was determined by staining cells with Annexin V-FITC (BD Pharmingen, US). The signals were detected by flow cytometry on a Beckman Coulter FC500. 2.6. Oxidative DNA damage Cells were treated with atovaquone for 24 h. Cells were harvested and DNA was extracted using the DNEasy Mini Kit (Qiagen). 8-Hydroxydeoxyguanosine (8-OHdG) levels were quantified using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to manufacture's protocol. Absorbance was read on a Spectramax M5 Microplate reader at 450 nm.
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2.7. Statistical analyses The data are obtained from at least three independent experiments and expressed as mean and standard deviation (SD). Statistical analyses were performed by unpaired Student's t-test, with p-value < 0.05 considered statistically significant. 3. Results 3.1. Atovaquone selectively targets malignant retinal cells and increases chemosensitivity Due to the inherent molecular complexity of retinoblastoma [4], we used three cell lines (RB116, Y79 and WERI-Rb1) presenting different cellular origin and genetic profiling of human retinoblastoma in our study. High plasma concentrations (40e80 mM) are routinely achieved in patients receiving atovaquone therapy [15]. We found that atovaquone at 5, 10 and 20 mM significantly inhibited proliferation and induces apoptosis of all tested retinoblastoma cell lines (Fig. 1A and B), demonstrating that the effective dose of atovaquone in retinoblastoma is clinically achievable. In contrast, atovaquone did not affect retinoblastoma cell migration (Fig. 1C). We next investigated the effects of atovaquone on healthy retinal cells using RPE-1 and HNRPE. RPE-1 is an immortalized normal retinal pigmented epithelial cell line and HNRPE is a primary normal retinal pigmented epithelial cell isolated from healthy
Fig. 2. Atovaquone disrupts mitochondrial function in both retinoblastoma and normal retinal cells. Atovaquone at 10 and 20 mM significantly decreases basal (A) and maximal (B) mitochondrial respiration in retinoblastoma cells: RB116, Y79 and WERI-Rb1 cells than normal retinal cells: RPE-1 and HNRPE. Atovaquone has no effect on mitochondrial membrane potential (C) and mass (D) in malignant and normal retinal cells. (E) Atovaquone significantly decreases ATP levels without affecting glycolysis (F) in malignant and normal retinal cells. *p < 0.05, compared to control.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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donor. We found that atovaquone at 10 and 20 mM significantly inhibited proliferation and induces apoptosis but not migration of normal retinal cells, with a less extent than in retinoblastoma cells (Fig. 1AeC). This indicates that atovaquone is more effective in targeting malignant than normal retinal cells. To further investigate the translational potential of atovaquone in retinoblastoma, we determined the combinatory effects of atovaquone with vincristine (a standard treatment for retinoblastoma). We found that the combinations of atovaquone with vincristine at different concentrations are all significantly more effective than vincristine or atovaquone alone in retinoblastoma cells (Fig. 1D and E). Notably, the combination almost induced complete apoptosis in retinoblastoma cells (Fig. 1E). These data clearly demonstrate that atovaquone increases chemosensitivity in retinoblastoma. 3.2. Atovaquone induces mitochondrial function in both normal and malignant retinal cells Atovaquone has been indicated to induce malarial parasite mitochondrial dysfunction via specifically targeting the cytochrome bc1 complex of the mitochondrial respiratory chain [16]. To examine whether atovaquone also targets mitochondria in retinoblastoma, we analyzed mitochondrial function and biogenesis in retinoblastoma cells after atovaquone treatment. We found that atovaquone at 10 and 20 mM significantly decreases basal and maximal mitochondrial respiration (indicated by oxygen consumption rate) without affecting mitochondrial membrane
potential and mass in retinoblastoma cells (Fig. 2AeD). As expected, ATP levels were significantly reduced in retinoblastoma cells exposed to atovaquone (Fig. 2E). Interestingly, it is noted that normal retinal cells have less mitochondrial respiration level, mitochondrial membrane potential, mass and ATP levels compared to retinoblastoma cells (Fig. 2AeE). In addition, atovaquone also decreases mitochondrial respiration and ATP levels without affecting mitochondrial membrane potential and mass in normal retinal cells (Fig. 2AeE). In contrast, there was no difference in glycolysis level between retinoblastoma and normal retinal cells (Fig. 2F). In addition, atovaquone did not affect glycolysis in both types of cells. This demonstrates that atovaquone targets malignant and normal retinal cells via the same mechanism. 3.3. Atovaquone induces oxidative stress and damage in retinoblastoma cells while normal retinal cells are largely unaffected Mitochondrial dysfunction is a major source of mitochondrial superoxide and intracellular ROS [17]. As expected, we found that atovaquone significantly increased mitochondrial superoxide and ROS levels in retinoblastoma cells (Fig. 3A and B). Consistently, oxidative DNA damage marker 8-Hydroxydeoxyguanosine (8-OHdG) was increased in retinoblastoma exposed to atovaquone (Fig. 3C). Atovaquone also significantly increased mitochondrial superoxide, ROS and 8-OHdG levels in normal retinal cells, but to a less extent than in retinoblastoma cells (Fig. 3AeC). These data indicate that atovaquone induces oxidative stress and damage in retinoblastoma cells while normal retinal cells are largely unaffected. Molecular
Fig. 3. Atovaquone induces oxidative stress and inhibits Akt/AMPK/mTOR in retinoblastoma and normal retinal cells. Atovaquone at 10 and 20 mM increases mitochondrial superoxide (A), intracellular ROS (B) and 8-OHdG (C) levels in malignant and normal retinal cells. (D) Atovaquone decreases p-Akt, p-mTOR and p-S6, and increases p-AMPK levels in RB116 and RPE cells. *p < 0.05, compared to control.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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analysis found that atovaquone inhibited phosphorylation of Akt and activated 50 -adenosine monophosphate-activated protein kinase (AMPK), leading to the inhibition of mammalian target of rapamycin (mTOR) signaling in retinoblastoma cells (Fig. 3D). 3.4. Mitochondrial respiration is required for the action of atovaquone in retinoblastoma To confirm mitochondria as the target of atovaquone in retinoblastoma, we examined the effects of atovaquone in mitochondrial respiration-deficient r0 cells. We successfully generated RB116 but not Y79 or WERI-Rb1 r0 by prolonged exposure of parental cells to ethidium bromide (a DNA intercalating agent) to remove mitochondrial DNA [18]. We confirmed that RB116 r0 was mitochondrial respiration deficient (Fig. 4A). In addition, we observed that RB116 r0 cells had remarkably slow growth rate and low ATP level compared to parental cells (Fig. 4B and C). We further observed that RB116 r0 cells had slightly but significantly higher levels of mitochondrial superoxide and intracellular ROS than parental cells (Fig. D and E). Importantly, we found that atovaquone is ineffective in decreasing ATP, inducing oxidative stress and damage, and inducing apoptosis in RB116 r0 cells (Fig. 4CeG). In addition, the inhibition of Akt/AMPK/mTOR pathway by atovaquone was reversed in RB116 r0 cells (Fig. 4H). Taken together, our results demonstrate that mitochondrial respiration is required for the action of atovaquone in retinoblastoma.
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4. Discussion One approach to develop therapies for cancer is to target common cancer drivers, particularly in cancers with extensive intraand inter-heterogeneity such as retinoblastoma. In this work, we demonstrate that the antimalarial atovaquone is active against retinoblastoma and increases chemosensitivity, while less toxicity to normal retinal cells. In addition, we provide detailed evidence that atovaquone acts by inhibiting mitochondrial function because of a dependence of retinoblastoma cells on this aspect of cell behavior. Atovaquone has been recently identified as a novel type of anticancer drug which is actively against various cancers including leukemia, anaplastic thyroid cancer and cervical cancer [19e21]. Using a panel of retinoblastoma cell lines that present different cellular origin and genetic background [22,23], we demonstrate that atovaquone at clinically achievable concentrations inhibited proliferation and induces apoptosis without affecting migration of all tested cells (Fig. 1AeC). These suggest the inhibitory effects of atovaquone on retinoblastoma development but not metastasis. In addition, the effective concentration range of atovaquone in retinoblastoma is consistent with the previous findings [19e21]. Our work adds retinoblastoma to the list of atovaquone-targeted cancers. Consistent with the previous work that atovaquone enhances doxorubin's efficacy in thyroid cancer [20], atovaquone significantly increases chemosensitivity in retinoblastoma (Fig. 1D and E). Our
Fig. 4. Atovaquone is ineffective in mitochondrial respiration-deficient r0 retinoblastoma cells. RB116 r0 cells has minimal level of mitochondrial respiration (A) and growth (B). Atovaquone is ineffective in decreasing ATP (C) and increasing mitoSox (D), ROS (E) and 8-OHdG (F) level and in RB116 r0 cells. (G) Atovaquone is ineffective in inducing apoptosis in RB116 r0 cells. (H) Atovaquone is ineffective in decreasing p-Akt, p-mTOR and p-S6 and increasing p-AMPK levels in RB116 r0 cells. Atovaquone at 20 mM was used. *p < 0.05, compared to RB116 cells.
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work together with others demonstrate the synergism between atovaquone and chemotherapeutic agents which suggests the translational potential of atovaquone in cancer treatment. Atovaquone is an attractive candidate for retinoblastoma as 1) it is available for clinical use in the treatment of broad spectrum of parasites [15]; 2) clinical trial demonstrates that atovaquone administration is associated with improved outcomes in patient with leukemia [19]; 3) effective concentrations routinely achieved in patients with well-tolerance. Besides gentle gastric discomfort (eg, diarrhoea and nausea) and rashes, atovaquone suspension is well tolerated [24]. The preferential selectivity of atovaquone on tumor rather than normal counterparts is demonstrated by our findings that atovaquone has much less toxicity effects to normal retinal cells (Fig. 1AeC) and previous work that atovaquone at similar concentrations is not toxic to fibroblast cells [25]. A significant finding of our work is that the preferential selectivity of atovaquone between malignant and normal retinal cells is due to their differential sensitivity to mitochondrial dysfunction (Fig. 2). Our findings support the previous work on the varying levels of mitochondrial biogenesis and response to mitochondrial inhibition between tumor and normal counterparts [9,26,27]. Using mitochondrial respiration-deficient r0 cells, we confirmed mitochondrial respiration as the target of atovaquone in retinoblastoma (Fig. 4). Targeting metabolic pathways for cancer therapy has attracted attention ever since Warburg's discovery of aerobic glycolysis [28]. However, our findings suggest that Warburg's paradigm of reprogramming energy metabolism may not necessarily apply to retinoblastoma as we only observed the differences on mitochondrial biogenesis but not glycolysis between retinoblastoma and normal retinal cells (Fig. 2). In addition, atovaquone specifically inhibits mitochondrial functions without affecting glycolysis (Fig. 2F). It is known that atovaquone kills parasites via inhibiting the cytochrome bc1 complex and mitochondrial respiration [12,13], suggesting that atovaquone acts on parasites and mammalian cells via a similar mechanism. As a consequence of mitochondrial dysfunction, our work provides clear details that oxidative stress and damage, and Akt/AMPK/mTOR inhibition is involved in the action of atovaquone in retinoblastoma. In conclusion, our work show that atovaquone exerts selective toxicity and increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/ AMPK/mTOR inhibition. Our work suggests that mitochondria is a selective target in retinoblastoma and awaits therapeutic exploitation. Conflicts of interest All authors declare no conflict of interest. Acknowledgement We acknowledge the many helpful comments and discussions with our colleagues. This work was supported by a research grant provided by Hubei University of Medicine (2015610185). References [1] L. Lumbroso-Le Rouic, A. Savignoni, C. Levy-Gabriel, I. Aerts, N. Cassoux, F. Salviat, M. Gauthier-Villars, P. Freneaux, H. Brisse, R. Dendale, M. Esteve, F. Doz, L. Desjardins, Treatment of retinoblastoma: the Institut Curie experience on a series of 730 patients (1995 to 2009), J. Fr. Ophtalmol. 38 (2015) 535e541. [2] S.H. Friend, R. Bernards, S. Rogelj, R.A. Weinberg, J.M. Rapaport, D.M. Albert, T.P. Dryja, A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma, Nature 323 (1986) 643e646. [3] K.A. Wikenheiser-Brokamp, Retinoblastoma family proteins: insights gained through genetic manipulation of mice, Cell. Mol. Life Sci. 63 (2006) 767e780.
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The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition Feng Ke a, Jinqiang Yu a, Wei Chen b, Xiaomin Si c, Xinhui Li d, Fang Yang a, Yingying Liao e, *, Zhigang Zuo f, ** a
Department of Ophthalmology, Renmin Hospital, Hubei University of Medicine, Shiyan, China Department of Critical Care Medicine, Taihe Hospital, Hubei University of Medicine, Hubei, China Department of Cardiopulmonary Rehabilitation, Renmin Hospital, Hubei University of Medicine, Shiyan, China d Department of Oncology, Shiyan Taihe Hospital, Shiyan, China e Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Shiyan, China f Department of Oncology, Renmin Hospital, Hubei University of Medicine, Shiyan, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2018 Accepted 10 June 2018 Available online xxx
Mitochondria has been identified as a promising target in several cancers. However, little is known on the effects of targeting mitochondria in retinoblastoma. In this work, we show that anti-malarial atovaquone, at clinically achievable concentration, demonstrates inhibitory effects to retinoblastoma cells, to a more extent than in normal retinal cells. Atovaquone also significantly increases chemosensitivity in retinoblastoma. Importantly, we show that retinoblastoma cells have higher level of mitochondrial respiration, membrane potential, mass and ATP compared to normal retinal cells. Although atovaquone significantly inhibits mitochondrial respiration and decrease ATP level in both malignant and normal retinal cells in a similar manner, atovaquone induces much more oxidative stress and damage in retinoblastoma than normal retinal cells. These suggest that normal retinal cells are more tolerable to mitochondrial dysfunctions than retinoblastoma cells. We further demonstrate that atovaquone targets Akt/AMPK/mTOR signaling via inducing mitochondrial dysfunction. Our pre-clinical work demonstrates the translational potential of atovaquone as an addition to the treatment armamentarium for retinoblastoma. Our work also demonstrates the differences of mitochondrial biogenesis and function in malignant versus normal retinal cells which are important for the targeted therapy in retinoblastoma. © 2018 Elsevier Inc. All rights reserved.
Keywords: Atovaquone Retinoblastoma Normal retina Mitochondria Akt/AMPK/mTOR
1. Introduction Retinoblastoma is an ocular cancer of retinal origin and typically occurs children younger than 5 years. Although the standard treatment including enucleation, chemotherapy and radiation significantly improves clinical outcome, the management of retinoblastoma is still challenge in developing countries [1]. Mutation of the tumor suppressor gene retinoblastoma 1 (RB1) plays a
* Corresponding author. Department of Gastroenterology, Renmin Hospital, Hubei University of Medicine, Maojian District, Chaoyangzhong Road 39, 442000, China. ** Corresponding author. Department of Oncology, Renmin Hospital, Hubei University of Medicine, Maojian District, Chaoyangzhong Road 39, 442000, China. E-mail addresses: [email protected] (Y. Liao), [email protected] (Z. Zuo).
central role in retinoblastoma transformation and progression [2,3]. Substantial evidence has shown extensive heterogeneity in retinoblastoma at molecular, cellular and tumor level [4], suggesting that targeting common cancer drivers may represent a potential therapeutic strategy for retinoblastoma. The mitochondria is an important energy and biosynthetic factories supporting cancer cell growth and survival [5]. Apart from energy metabolism, other functions of mitochondria include calcium homeostasis, redox regulation and apoptosis [6]. Importantly, studies have recently shown that cancer cells have increased mitochondrial biogenesis and rely more on mitochondrial respiration compared to normal cells [7e9]. Inhibition of mitochondrial functions display anti-cancer activities while having less toxicity to normal counterparts [10]. Targeting mitochondria using clinically available drugs as a cancer therapeutic strategy has attracted much
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Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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attention in the recent years and shown translational potential in pre-clinical and clinical settings [8,11]. Atovaquone is an anti-malarial drug with its target on cytochrome bc1 complex and therefore affecting mitochondrial function [12,13]. In this study, we investigated the effect and target of atovaquone in retinoblastoma (malignant) and normal retinal cells. We further investigated the basal mitochondrial biogenesis of malignant and normal retinal cells to understand their differential response to atovaquone. Our findings demonstrate that inhibition of mitochondrial respiration as a potential targeted strategy for retinoblastoma. 2. Materials and methods 2.1. Primary cells, cell culture and generation of r0 cell line Human retinoblastoma cell lines RB116 (Kerafast Inc. US), Y79 and WERI-Rb-1 (ATCC, US) and immortalized normal retinoblastoma pigmented epithelial cell line (RPE-1, ATCC, US) were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, and 4.5 g/L glucose. Primary normal retinoblastoma pigmented epithelial cell (HNRPE, Abm, US) was grown in extracellular matrix (Abm, US)-coated flask using the media (Abm, Cat. No. TM4057) according to manufacture's instruction. Mitochondria DNA-deficient r0 cell line was established according to the method previously described [14]. The lack of mitochondrial DNA in r0 cells was confirmed using reverse transcriptionePCR (data not shown).
2.2. Migration assays Migration assay was performed using the Boyden chamber which consists a Falcon cell culture insert and 24-well plate chamber. Cells (10,000/well) and atovaquone were added into the cell culture insert. 20% FBS was placed into the lower chamber. The assembled cell culture insert chamber was incubated for 6e8 h. Cells on the upper surface of the insert were then removed with a cotton swab. Migratory cells on the lower surface of inserts were fixed with 4% formaldehyde (Sigma, US) for 10 min, then stained with 0.4% Giemsa and counted under using light microscope (Zeiss, Germany). 2.3. Proliferation assay Cells (10, 000/well) were treated with atovaquone, vincristine or combination for 72 h in 96-well-plate. Proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation assay kit (Promega, US). 2.4. Mito stress and glycolytic stress test assays Cells (10,000/well) were seeded, cultured and treated with atovaquone in XF96 cell culture plates. After treatment, media were replaced by XF assay medium (Seahorse Bioscience, US) and incubated at 37 C in a CO2-free environment for 1 h. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at 37 C using an XF96 extracellular analyzer
Fig. 1. Atovaquone selectively targets retinoblastoma cells and enhances the inhibitory effects of chemotherapeutic agent. Atovaquone is more effective in inhibiting proliferation (A) and inducing apoptosis (B) in retinoblastoma cells: RB116, Y79 and WERI-Rb1 cells than normal retinal cells: RPE-1 and HNRPE. Atovaquone significantly enhances the anti-proliferative (C) and pro-apoptotic (D) effects of vincristine in retinoblastoma cells. Atovaquone at 5 mM was used in combination studies. *p < 0.05, compared to control or vincristine alone.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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(Seahorse Bioscience, US) as per the manufacture's instructions. All injection reagents (as detailed in the figure legends) were adjusted to pH 7.4. The Seahorse software calculated OCR and ECAR automatically. 2.5. Flow cytometry Cells (50, 000/well) were treated with atovaquone in 24-wellplate for 24 h. Cells were stained with CM-H2DCFDA (indicates intracellular ROS), MitoSox Red (indicates mitochondrial superoxide), MitoTracker (indicates mitochondrial mass), or 5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethyl benzimidazolylcarbocyanine iodide (JC-1 to indicate mitochondrial membrane potential) as per manufacture's instructions (Invitrogen, US). Apoptosis was determined by staining cells with Annexin V-FITC (BD Pharmingen, US). The signals were detected by flow cytometry on a Beckman Coulter FC500. 2.6. Oxidative DNA damage Cells were treated with atovaquone for 24 h. Cells were harvested and DNA was extracted using the DNEasy Mini Kit (Qiagen). 8-Hydroxydeoxyguanosine (8-OHdG) levels were quantified using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) according to manufacture's protocol. Absorbance was read on a Spectramax M5 Microplate reader at 450 nm.
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2.7. Statistical analyses The data are obtained from at least three independent experiments and expressed as mean and standard deviation (SD). Statistical analyses were performed by unpaired Student's t-test, with p-value < 0.05 considered statistically significant. 3. Results 3.1. Atovaquone selectively targets malignant retinal cells and increases chemosensitivity Due to the inherent molecular complexity of retinoblastoma [4], we used three cell lines (RB116, Y79 and WERI-Rb1) presenting different cellular origin and genetic profiling of human retinoblastoma in our study. High plasma concentrations (40e80 mM) are routinely achieved in patients receiving atovaquone therapy [15]. We found that atovaquone at 5, 10 and 20 mM significantly inhibited proliferation and induces apoptosis of all tested retinoblastoma cell lines (Fig. 1A and B), demonstrating that the effective dose of atovaquone in retinoblastoma is clinically achievable. In contrast, atovaquone did not affect retinoblastoma cell migration (Fig. 1C). We next investigated the effects of atovaquone on healthy retinal cells using RPE-1 and HNRPE. RPE-1 is an immortalized normal retinal pigmented epithelial cell line and HNRPE is a primary normal retinal pigmented epithelial cell isolated from healthy
Fig. 2. Atovaquone disrupts mitochondrial function in both retinoblastoma and normal retinal cells. Atovaquone at 10 and 20 mM significantly decreases basal (A) and maximal (B) mitochondrial respiration in retinoblastoma cells: RB116, Y79 and WERI-Rb1 cells than normal retinal cells: RPE-1 and HNRPE. Atovaquone has no effect on mitochondrial membrane potential (C) and mass (D) in malignant and normal retinal cells. (E) Atovaquone significantly decreases ATP levels without affecting glycolysis (F) in malignant and normal retinal cells. *p < 0.05, compared to control.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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donor. We found that atovaquone at 10 and 20 mM significantly inhibited proliferation and induces apoptosis but not migration of normal retinal cells, with a less extent than in retinoblastoma cells (Fig. 1AeC). This indicates that atovaquone is more effective in targeting malignant than normal retinal cells. To further investigate the translational potential of atovaquone in retinoblastoma, we determined the combinatory effects of atovaquone with vincristine (a standard treatment for retinoblastoma). We found that the combinations of atovaquone with vincristine at different concentrations are all significantly more effective than vincristine or atovaquone alone in retinoblastoma cells (Fig. 1D and E). Notably, the combination almost induced complete apoptosis in retinoblastoma cells (Fig. 1E). These data clearly demonstrate that atovaquone increases chemosensitivity in retinoblastoma. 3.2. Atovaquone induces mitochondrial function in both normal and malignant retinal cells Atovaquone has been indicated to induce malarial parasite mitochondrial dysfunction via specifically targeting the cytochrome bc1 complex of the mitochondrial respiratory chain [16]. To examine whether atovaquone also targets mitochondria in retinoblastoma, we analyzed mitochondrial function and biogenesis in retinoblastoma cells after atovaquone treatment. We found that atovaquone at 10 and 20 mM significantly decreases basal and maximal mitochondrial respiration (indicated by oxygen consumption rate) without affecting mitochondrial membrane
potential and mass in retinoblastoma cells (Fig. 2AeD). As expected, ATP levels were significantly reduced in retinoblastoma cells exposed to atovaquone (Fig. 2E). Interestingly, it is noted that normal retinal cells have less mitochondrial respiration level, mitochondrial membrane potential, mass and ATP levels compared to retinoblastoma cells (Fig. 2AeE). In addition, atovaquone also decreases mitochondrial respiration and ATP levels without affecting mitochondrial membrane potential and mass in normal retinal cells (Fig. 2AeE). In contrast, there was no difference in glycolysis level between retinoblastoma and normal retinal cells (Fig. 2F). In addition, atovaquone did not affect glycolysis in both types of cells. This demonstrates that atovaquone targets malignant and normal retinal cells via the same mechanism. 3.3. Atovaquone induces oxidative stress and damage in retinoblastoma cells while normal retinal cells are largely unaffected Mitochondrial dysfunction is a major source of mitochondrial superoxide and intracellular ROS [17]. As expected, we found that atovaquone significantly increased mitochondrial superoxide and ROS levels in retinoblastoma cells (Fig. 3A and B). Consistently, oxidative DNA damage marker 8-Hydroxydeoxyguanosine (8-OHdG) was increased in retinoblastoma exposed to atovaquone (Fig. 3C). Atovaquone also significantly increased mitochondrial superoxide, ROS and 8-OHdG levels in normal retinal cells, but to a less extent than in retinoblastoma cells (Fig. 3AeC). These data indicate that atovaquone induces oxidative stress and damage in retinoblastoma cells while normal retinal cells are largely unaffected. Molecular
Fig. 3. Atovaquone induces oxidative stress and inhibits Akt/AMPK/mTOR in retinoblastoma and normal retinal cells. Atovaquone at 10 and 20 mM increases mitochondrial superoxide (A), intracellular ROS (B) and 8-OHdG (C) levels in malignant and normal retinal cells. (D) Atovaquone decreases p-Akt, p-mTOR and p-S6, and increases p-AMPK levels in RB116 and RPE cells. *p < 0.05, compared to control.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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analysis found that atovaquone inhibited phosphorylation of Akt and activated 50 -adenosine monophosphate-activated protein kinase (AMPK), leading to the inhibition of mammalian target of rapamycin (mTOR) signaling in retinoblastoma cells (Fig. 3D). 3.4. Mitochondrial respiration is required for the action of atovaquone in retinoblastoma To confirm mitochondria as the target of atovaquone in retinoblastoma, we examined the effects of atovaquone in mitochondrial respiration-deficient r0 cells. We successfully generated RB116 but not Y79 or WERI-Rb1 r0 by prolonged exposure of parental cells to ethidium bromide (a DNA intercalating agent) to remove mitochondrial DNA [18]. We confirmed that RB116 r0 was mitochondrial respiration deficient (Fig. 4A). In addition, we observed that RB116 r0 cells had remarkably slow growth rate and low ATP level compared to parental cells (Fig. 4B and C). We further observed that RB116 r0 cells had slightly but significantly higher levels of mitochondrial superoxide and intracellular ROS than parental cells (Fig. D and E). Importantly, we found that atovaquone is ineffective in decreasing ATP, inducing oxidative stress and damage, and inducing apoptosis in RB116 r0 cells (Fig. 4CeG). In addition, the inhibition of Akt/AMPK/mTOR pathway by atovaquone was reversed in RB116 r0 cells (Fig. 4H). Taken together, our results demonstrate that mitochondrial respiration is required for the action of atovaquone in retinoblastoma.
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4. Discussion One approach to develop therapies for cancer is to target common cancer drivers, particularly in cancers with extensive intraand inter-heterogeneity such as retinoblastoma. In this work, we demonstrate that the antimalarial atovaquone is active against retinoblastoma and increases chemosensitivity, while less toxicity to normal retinal cells. In addition, we provide detailed evidence that atovaquone acts by inhibiting mitochondrial function because of a dependence of retinoblastoma cells on this aspect of cell behavior. Atovaquone has been recently identified as a novel type of anticancer drug which is actively against various cancers including leukemia, anaplastic thyroid cancer and cervical cancer [19e21]. Using a panel of retinoblastoma cell lines that present different cellular origin and genetic background [22,23], we demonstrate that atovaquone at clinically achievable concentrations inhibited proliferation and induces apoptosis without affecting migration of all tested cells (Fig. 1AeC). These suggest the inhibitory effects of atovaquone on retinoblastoma development but not metastasis. In addition, the effective concentration range of atovaquone in retinoblastoma is consistent with the previous findings [19e21]. Our work adds retinoblastoma to the list of atovaquone-targeted cancers. Consistent with the previous work that atovaquone enhances doxorubin's efficacy in thyroid cancer [20], atovaquone significantly increases chemosensitivity in retinoblastoma (Fig. 1D and E). Our
Fig. 4. Atovaquone is ineffective in mitochondrial respiration-deficient r0 retinoblastoma cells. RB116 r0 cells has minimal level of mitochondrial respiration (A) and growth (B). Atovaquone is ineffective in decreasing ATP (C) and increasing mitoSox (D), ROS (E) and 8-OHdG (F) level and in RB116 r0 cells. (G) Atovaquone is ineffective in inducing apoptosis in RB116 r0 cells. (H) Atovaquone is ineffective in decreasing p-Akt, p-mTOR and p-S6 and increasing p-AMPK levels in RB116 r0 cells. Atovaquone at 20 mM was used. *p < 0.05, compared to RB116 cells.
Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049
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work together with others demonstrate the synergism between atovaquone and chemotherapeutic agents which suggests the translational potential of atovaquone in cancer treatment. Atovaquone is an attractive candidate for retinoblastoma as 1) it is available for clinical use in the treatment of broad spectrum of parasites [15]; 2) clinical trial demonstrates that atovaquone administration is associated with improved outcomes in patient with leukemia [19]; 3) effective concentrations routinely achieved in patients with well-tolerance. Besides gentle gastric discomfort (eg, diarrhoea and nausea) and rashes, atovaquone suspension is well tolerated [24]. The preferential selectivity of atovaquone on tumor rather than normal counterparts is demonstrated by our findings that atovaquone has much less toxicity effects to normal retinal cells (Fig. 1AeC) and previous work that atovaquone at similar concentrations is not toxic to fibroblast cells [25]. A significant finding of our work is that the preferential selectivity of atovaquone between malignant and normal retinal cells is due to their differential sensitivity to mitochondrial dysfunction (Fig. 2). Our findings support the previous work on the varying levels of mitochondrial biogenesis and response to mitochondrial inhibition between tumor and normal counterparts [9,26,27]. Using mitochondrial respiration-deficient r0 cells, we confirmed mitochondrial respiration as the target of atovaquone in retinoblastoma (Fig. 4). Targeting metabolic pathways for cancer therapy has attracted attention ever since Warburg's discovery of aerobic glycolysis [28]. However, our findings suggest that Warburg's paradigm of reprogramming energy metabolism may not necessarily apply to retinoblastoma as we only observed the differences on mitochondrial biogenesis but not glycolysis between retinoblastoma and normal retinal cells (Fig. 2). In addition, atovaquone specifically inhibits mitochondrial functions without affecting glycolysis (Fig. 2F). It is known that atovaquone kills parasites via inhibiting the cytochrome bc1 complex and mitochondrial respiration [12,13], suggesting that atovaquone acts on parasites and mammalian cells via a similar mechanism. As a consequence of mitochondrial dysfunction, our work provides clear details that oxidative stress and damage, and Akt/AMPK/mTOR inhibition is involved in the action of atovaquone in retinoblastoma. In conclusion, our work show that atovaquone exerts selective toxicity and increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/ AMPK/mTOR inhibition. Our work suggests that mitochondria is a selective target in retinoblastoma and awaits therapeutic exploitation. Conflicts of interest All authors declare no conflict of interest. Acknowledgement We acknowledge the many helpful comments and discussions with our colleagues. This work was supported by a research grant provided by Hubei University of Medicine (2015610185). References [1] L. Lumbroso-Le Rouic, A. Savignoni, C. Levy-Gabriel, I. Aerts, N. Cassoux, F. Salviat, M. Gauthier-Villars, P. Freneaux, H. Brisse, R. Dendale, M. Esteve, F. Doz, L. Desjardins, Treatment of retinoblastoma: the Institut Curie experience on a series of 730 patients (1995 to 2009), J. Fr. Ophtalmol. 38 (2015) 535e541. [2] S.H. Friend, R. Bernards, S. Rogelj, R.A. Weinberg, J.M. Rapaport, D.M. Albert, T.P. Dryja, A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma, Nature 323 (1986) 643e646. [3] K.A. Wikenheiser-Brokamp, Retinoblastoma family proteins: insights gained through genetic manipulation of mice, Cell. Mol. Life Sci. 63 (2006) 767e780.
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Please cite this article in press as: F. Ke, et al., The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.049